High-throughput screening for ligands of transmembrane proteins

Abstract

A high-throughput screening for at least one ligand of at least one transmembrane protein (TP) of interest that is embedded in a lipid bilayer of at least one endogenous microsome derived from plant-based endoplasmatic reticulum (ER). The screening includes providing at least one endogenous microsome that includes at least one lipid bilayer embedded TP or at least one endogenous microsomal fragment that includes at least one lipid bilayer embedded TP, and providing at least one analyte of interest, contacting of the at least one TP with the at least one analyte and detection of interaction between the at least one analyte and the at least one TP. The screening is suitable for high multiplex grades of TPs and analytes and for a fast and reproducible identification of ligands as potential drug candidates. All essential products, consumables and kits for use in the high-throughput screening are described.

1 . A high-throughput screening for at least one ligand of at least one transmembrane protein (TP) of interest that is embedded in a lipid bilayer of at least one endogenous microsome derived from plant-based endoplasmatic reticulum (ER), comprising:
 providing at least one endogenous microsome comprising at least one lipid bilayer embedded transmembrane protein (TP) or at least one endogenous microsomal fragment comprising at least one lipid bilayer embedded transmembrane protein (TP),   providing at least one analyte of interest,   contacting of the at least one TP with the at least one analyte and   detection of interaction between the at least one analyte and the at least one TP.   
     
     
         2 . The screening according to  claim 1 , wherein the at least one microsome or microsomal fragment is derived from the genus  Nicotiana  of the family Solanaceae. 
     
     
         3 . The screening according to  claim 1 , further comprising capturing of the at least one TP embedded within a lipid bilayer of a microsome or a microsomal fragment to a biologically non-active surface. 
     
     
         4 . The screening according to  claim 1 , wherein the at least one TP embedded in a lipid bilayer of the endogenous microsome and/or the endogenous microsomal fragment is captured on a biologically non-active surface. 
     
     
         5 . The screening according to  claim 1 , wherein the biologically non-active surface is a surface of a particle comprising micro-particles, nano-particles and magnetic particles, a surface of a device comprising microtiter plates, microfluidic devices, micro arrays, microchips and/or it is any other surface suitable for capturing the at least one TP of interest. 
     
     
         6 . The screening according to  claim 1 , wherein the at least one TP comprises at least one tag at the C-terminus and/or N-terminus. 
     
     
         7 . The screening according to  claim 1 , wherein the at least one analyte comprises a small molecule, peptide, polypeptide, soluble protein, membrane protein, protein complex or any combination of the aforementioned. 
     
     
         8 . The screening according to  claim 1 , wherein it is a multiplex High-throughput screening for at least one ligand of at least one TP of interest providing two or more analytes in two or more reaction zones simultaneously and/or providing an analyte combination of two or more analytes simultaneously in two or more reaction zones. 
     
     
         9 . The screening according to  claim 1 , wherein the interaction between the at least one TP and the at least one analyte is detected by an immunoassay, a spectroscopic assay, a particle size measurement, a magnetic measurement and/or an optical measurement. 
     
     
         10 . The screening according to  claim 1 , wherein the least one endogenous microsome or endogenous microsomal fragment derived from plant-based endoplasmatic reticulum is from a cellular lysate from a plant of the genus  Nicotiana  of the family Solanaceae. 
     
     
         11 . The screening according to  claim 1 , wherein the lysate is from a BY-2 cell line from  Nicotiana tabacum.    
     
     
         12 . A cell-free production of at least one transmembrane protein (TP) of interest that is embedded in a lipid bilayer of at least one endogenous microsome derived from plant-based endoplasmatic reticulum (ER), comprising:
 providing a lysate from a plant comprising at least one endogenous microsome   providing at least one template encoding for the at least one transmembrane protein (TP),   incubation of the at least one lysate with the at least one template,   expression of the at least one transmembrane protein (TP),   coupled transcription and co-translational translocation of the at least one expressed TP into the at least one microsome and   obtaining the at least one transmembrane protein (TP) embedded in a lipid bilayer of the at least one endogenous microsome and   optionally treating the at least one endogenous microsome comprising the at least one TP embedded in a lipid bilayer to obtain microsomal fragments comprising the at least one TP embedded in a lipid bilayer.   
     
     
         13 . The method of  claim 12 , further comprising capturing of the at least one TP embedded within a lipid bilayer of a microsome on the at least one biologically non-active surface. 
     
     
         14 . The method of  claim 13 , wherein the capturing is performed and treating the at least one captured microsome is performed to obtain microsomal fragments, wherein the at least one TP remains embedded in a lipid bilayer. 
     
     
         15 . The method of  claim 13 , wherein treating of the at least one free microsome is performed to obtain free microsomal fragments, wherein the at least one TP remains embedded in a lipid bilayer, and capturing of the at least one TP embedded within a lipid bilayer of the microsomal fragment on the at least one biologically non active surface is performed. 
     
     
         16 . The method of  claim 13 , wherein the biologically non-active surface is a surface of a particle comprising micro-particles, nano-particles and magnetic particles, a surface of a device comprising microtiter plates, microfluidic devices, micro arrays, microchips and/or it is any other surface suitable for capturing the at least one TP of interest. 
     
     
         17 . The method according to  claim 12 , wherein the at least one microsome or microsomal fragment is derived from the genus  Nicotiana  of the family Solanaceae. 
     
     
         18 . An endogenous microsome or at least one endogenous microsomal fragment derived from plant-based endoplasmatic reticulum (ER) comprising at least one lipid bilayer embedded transmembrane protein (TP) of interest. 
     
     
         19 . A biological non active surface comprising at least one captured transmembrane protein (TP) of interest that is embedded in a lipid bilayer of at least one endogenous microsome or of at least one endogenous microsomal fragment derived from plant-based endoplasmatic reticulum (ER), wherein the biologically non-active surface is a surface of a consumable. 
     
     
         20 . A kit for a high-throughput screening for at least one ligand of at least one transmembrane protein (TP) of interest that is embedded in a lipid bilayer of at least one endogenous microsome derived from plant-based endoplasmatic reticulum (ER), preferably derived from a plant of the genus  Nicotiana  of the family Solanaceae comprising
 a plant cellular lysate comprising endoplasmatic reticulum (ER) derived endogenous microsomes preferably from a plant of the genus  Nicotiana  of the family Solanaceae,   at least one vector encoding for a known TP as positive control   at least one vector for at least one template encoding for the at least one TP of interest   optionally further excipients,   optionally at least one agent for detection of the interaction between the at least one analyte and the at least one TP,   optionally at least one agent for destruction of the at least one microsome and   optionally at least one analyte or an analyte panel of two or more analytes.   
     
     
         21 . The kit of  claim 20 , further comprising a consumable that comprises, consists of or is coated with a biological non-active surface for capturing of the at least one TP embedded within a lipid bilayer of a microsome to said biologically non-active surface.